title: The proteases HtrA2/Omi and UCH-L1 regulate TNF-induced necroptosis.
abstract: BACKGROUND
  In apoptosis, proteolysis by caspases is the primary mechanism for both initiation and execution of programmed cell death (PCD). In contrast, the impact of proteolysis on the regulation and execution of caspase-independent forms of PCD (programmed necrosis, necroptosis) is only marginally understood. Likewise, the identity of the involved proteases has remained largely obscure. Here, we have investigated the impact of proteases in TNF-induced necroptosis.
  
  
  RESULTS
  The serine protease inhibitor TPKC protected from TNF-induced necroptosis in multiple murine and human cells systems whereas inhibitors of metalloproteinases or calpain/cysteine and cathepsin proteases had no effect. A screen for proteins labeled by a fluorescent TPCK derivative in necroptotic cells identified HtrA2/Omi (a serine protease previously implicated in PCD) as a promising candidate. Demonstrating its functional impact, pharmacological inhibition or genetic deletion of HtrA2/Omi protected from TNF-induced necroptosis. Unlike in apoptosis, HtrA2/Omi did not cleave another protease, ubiquitin C-terminal hydrolase (UCH-L1) during TNF-induced necroptosis, but rather induced monoubiquitination indicative for UCH-L1 activation. Correspondingly, pharmacologic or RNA interference-mediated inhibition of UCH-L1 protected from TNF-induced necroptosis. We found that UCH-L1 is a mediator of caspase-independent, non-apoptotic cell death also in diseased kidney podocytes by measuring cleavage of the protein PARP-1, caspase activity, cell death and cell morphology. Indicating a role of TNF in this process, podocytes with stably downregulated UCH-L1 proved resistant to TNF-induced necroptosis.
  
  
  CONCLUSIONS
  The proteases HtrA2/Omi and UCH-L1 represent two key components of TNF-induced necroptosis, validating the relevance of proteolysis not only for apoptosis, but also for caspase-independent PCD. Since UCH-L1 clearly contributes to the non-apoptotic death of podocytes, interference with the necroptotic properties of HtrA2/Omi and UCH-L1 may prove beneficial for the treatment of patients, e.g. in kidney failure.
authors.affiliation: Institut für Immunologie, Christian-Albrechts-Universität zu Kiel, Michaelisstr, 5, 24105 Kiel, Germany. dadam@email.uni-kiel.de.
authors.forename: Justyna
authors.initials: J
authors.lastname: Sosna
authors.forename: Susann
authors.initials: S
authors.lastname: Voigt
authors.forename: Sabine
authors.initials: S
authors.lastname: Mathieu
authors.forename: Dieter
authors.initials: D
authors.lastname: Kabelitz
authors.forename: Ahmad
authors.initials: A
authors.lastname: Trad
authors.forename: Ottmar
authors.initials: O
authors.lastname: Janssen
authors.forename: Catherine
authors.initials: C
authors.lastname: Meyer-Schwesinger
authors.forename: Stefan
authors.initials: S
authors.lastname: Schütze
authors.forename: Dieter
authors.initials: D
authors.lastname: Adam
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002478:Cells, Cultured; D019073:HT29 Cells; D000074923:High-Temperature Requirement A Serine Peptidase 2; D006801:Humans; D019169:Jurkat Cells; D051379:Mice; D024101:Mitochondrial Proteins / Q000378:metabolism*; D041681:NIH 3T3 Cells; D050199:Podocytes / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D012697:Serine Endopeptidases / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*; D043222:Ubiquitin Thiolesterase / Q000378:metabolism*
chemical_list: D000074923:High-Temperature Requirement A Serine Peptidase 2; D024101:Mitochondrial Proteins; D012697:Serine Endopeptidases; D014409:Tumor Necrosis Factor-alpha; D043222:Ubiquitin Thiolesterase; C482860:UCHL1 protein, human; C489034:UCHL1 protein, rat; C000615914:HTRA2 protein, human; C000615915:Htra2 protein, mouse; C496976:Uchl1 protein, mouse